Identification and selection of optimal reference genes for qPCR-based gene expression analysis in Fucus distichus under various abiotic stresses

Quantitative gene expression analysis is an important tool in the scientist’s belt. The identification of evenly expressed reference genes is necessary for accurate quantitative gene expression analysis, whether by traditional RT-PCR (reverse-transcription polymerase chain reaction) or by qRT-PCR (quantitative real-time PCR; qPCR). In the Stramenopiles (the major line of eukaryotes that includes brown algae) there is a noted lack of known reference genes for such studies, largely due to the absence of available molecular tools. Here we present a set of nine reference genes (Elongation Factor 1 alpha (EF1A), Elongation Factor 2 alpha (EF2A), Elongation Factor 1 beta (EF1B), 14-3-3 Protein, Ubiquitin Conjugating Enzyme (UBCE2), Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH), Actin Related Protein Complex (ARP2/3), Ribosomal Protein (40s; S23), and Actin) for the brown alga Fucus distichus. These reference genes were tested on adult sporophytes across six abiotic stress conditions (desiccation, light and temperature modification, hormone addition, pollutant exposure, nutrient addition, and wounding). Suitability of these genes as reference genes was quantitatively evaluated across conditions using standard methods and the majority of the tested genes were evaluated favorably. However, we show that normalization genes should be chosen on a condition-by-condition basis. We provide a recommendation that at least two reference genes be used per experiment, a list of recommended pairs for the conditions tested here, and a procedure for identifying a suitable set for an experimenter’s unique design. With the recent expansion of interest in brown algal biology and accompanied molecular tools development, the variety of experimental conditions tested here makes this study a valuable resource for future work in basic biology and understanding stress responses in the brown algal lineage.

Characterisation and chemometric evaluation of 17 elements in ten seaweed species from Greenland

Several Greenland seaweed species have potential as foods or food ingredients, both for local consumption and export. However, knowledge regarding their content of beneficial and deleterious elements on a species specific and geographical basis is lacking. This study investigated the content of 17 elements (As, Ca, Cd, Cr, Cu, Fe, Hg, I, K, Mg, Mn, Na, Ni, P, Pb, Se and Zn) in 77 samples of ten species (Agarum clathratum, Alaria esculenta, Ascophyllum nodosum, Fucus distichus, Fucus vesiculosus, Hedophyllum nigripes, Laminaria solidungula, Palmaria palmata, Saccharina latissima and Saccharina longicruris). Element profiles differed between species but showed similar patterns within the same family. For five species, different thallus parts were investigated separately, and showed different element profiles. A geographic origin comparison of Fucus species indicated regional differences. The seaweeds investigated were especially good sources of macrominerals (K > Na > Ca > Mg) and trace minerals, such as Fe. Iodine contents were high, especially in macroalgae of the family Laminariaceae. None of the samples exceeded the EU maximum levels for Cd, Hg or Pb, but some exceeded the stricter French regulations, especially for Cd and I. In conclusion, these ten species are promising food items.

Characterisation and chemometric evaluation of 17 elements in ten seaweed species from Greenland

Several Greenland seaweed species have potential as foods or food ingredients, both for local consumption and export. However, knowledge regarding their content of beneficial and deleterious elements on a species specific and geographical basis is lacking. This study investigated the content of 17 elements (As, Ca, Cd, Cr, Cu, Fe, Hg, I, K, Mg, Mn, Na, Ni, P, Pb, Se and Zn) in 77 samples of ten species (Agarum clathratum, Alaria esculenta, Ascophyllum nodosum, Fucus distichus, Fucus vesiculosus, Hedophyllum nigripes, Laminaria solidungula, Palmaria palmata, Saccharina latissima and Saccharina longicruris). Element profiles differed between species but showed similar patterns within the same family. For five species, different thallus parts were investigated separately, and showed different element profiles. A geographic origin comparison of Fucus species indicated regional differences. The seaweeds investigated were especially good sources of macrominerals (K > Na > Ca > Mg) and trace minerals, such as Fe. Iodine contents were high, especially in macroalgae of the family Laminariaceae. None of the samples exceeded the EU maximum levels for Cd, Hg or Pb, but some exceeded the stricter French regulations, especially for Cd and I. In conclusion, these ten species are promising food items.

Enzyme-Assisted Fucoidan Extraction from Brown Macroalgae Fucus distichus subsp. evanescens and Saccharina latissima

Fucoidans from brown macroalgae (brown seaweeds) have different structures and many interesting bioactivities. Fucoidans are classically extracted from brown seaweeds by hot acidic extraction. Here, we report a new targeted enzyme-assisted methodology for fucoidan extraction from brown seaweeds. This enzyme-assisted extraction protocol involves a one-step combined use of a commercial cellulase preparation (Cellic®CTec2) and an alginate lyase from Sphingomonas sp. (SALy), reaction at pH 6.0, 40 °C, removal of non-fucoidan polysaccharides by Ca2+ precipitation, and ethanol-precipitation of crude fucoidan. The workability of this method is demonstrated for fucoidan extraction from Fucus distichus subsp. evanescens (basionym Fucus evanescens) and Saccharina latissima as compared with mild acidic extraction. The crude fucoidans resulting directly from the enzyme-assisted method contained considerable amounts of low molecular weight alginate, but this residual alginate was effectively removed by an additional ion-exchange chromatographic step to yield pure fucoidans (as confirmed by 1H NMR). The fucoidan yields that were obtained by the enzymatic method were comparable to the chemically extracted yields for both F. evanescens and S. latissima, but the molecular sizes of the fucoidans were significantly larger with enzyme-assisted extraction. The molecular weight distribution of the fucoidan fractions was 400 to 800 kDa for F. evanescens and 300 to 800 kDa for S. latissima, whereas the molecular weights of the corresponding chemically extracted fucoidans from these seaweeds were 10–100 kDa and 50–100 kDa, respectively. Enzyme-assisted extraction represents a new gentle strategy for fucoidan extraction and it provides new opportunities for obtaining high yields of native fucoidan structures from brown macroalgae.